1. Field of Invention
This invention relates to novel two-step warming protocol for warming cryopreserved cells from a cryopreservation temperature. The invention also relates to the use of a heat sink in the warming of the cryopreserved cells.
2. Description of Related Art
Cryobiology may be defined as the study of the effects of temperatures of lower than normal physiologic ranges upon biologic systems. During the past half-century the fundamentals of the science of cryobiology have evolved to the point where low temperatures are now used extensively as a means to protect and preserve biological systems during enforced periods of ischemia and hypoxia. In practice, preservation is achieved using either hypothermia without freezing, or cryopreservation in which the aqueous system sustains a physical phase change with the formation of ice. Survival of cells from the rigors of freezing and thawing in cryopreservation procedures is only attained by using appropriate cryoprotective agents (CPAs) and in general, these techniques are applicable to isolated cells in suspension or small aggregates of cells in simple tissues. More complex tissues and organs having a defined architecture are not easily preserved using conventional cryopreservation techniques, which is principally due to the deleterious effects of ice formation in an organized multicellular tissue. Simply freezing cells or tissues results in dead, nonfunctional materials.
The modern era of cryobiology really began with the discovery of the cryoprotective properties of glycerol as reported by Polge et al., xe2x80x9cRevival of Spermatazoa After Vitrification and Dehydration at Low Temperatures,xe2x80x9d Nature, 164:666 (1949). Subsequently, Lovelock et al., xe2x80x9cPrevention of Freezing Damage to Living Cells by Dimethyl Sulfoxide,xe2x80x9d Nature, 183:1394 (1959), discovered that dimethyl sulfoxide was also a cryoprotectant, and despite the wide range of compounds now known to exhibit cryoprotective properties, it is still the most widely used compound to date.
A review of the principles of cryobiology can be found in Brockbank, Principles of Cryopreserved Venous Transplantation, Chapter 10, xe2x80x9cEssentials of Cryobiologyxe2x80x9d (1995). A basic principle of cryobiology is that the extent of freezing damage depends upon the amount of free water in the system and the ability of that water to crystallize during freezing. Many types of isolated cells and small aggregates of cells can be frozen simply by following published procedures, but obtaining reproducible results for more complex tissues requires an understanding of the major variables involved in tissue cryopreservation. Major variables involved in tissue freezing include (1) freezing-compatible pH buffers, (2) cryoprotectant choice, concentration and administration, (3) cooling protocol, (4) storage temperature, (5) warming protocol and (6) cryoprotectant elution.
Most research in cryobiology has focused upon finding and testing new types of cryoprotectants. Many cryoprotectants have been discovered. See, for example, Brockbank, supra. Freezing protocols for placing cells in cryopreservation and warming protocols for removing cryopreserved cells from cryopreservation are presently fairly standardized in the art.
However, the present inventors believe that the existing one-step warming protocols may contribute to losses of cells upon warming from the cryopreserved state, particularly with respect to cells attached to a fixed substrate. What is desired is an improved procedure for warming cryopreserved cells, particularly cells fixed to an attached substrate, from the frozen state so as to achieve an increase in the number of viable cells recovered from cryopreservation.
It is therefore one object of the present invention to provide a novel procedure for warming cryopreserved cells from a cryopreserved state that minimizes loss of cryopreserved cells.
These and other objects are achieved by the present invention, which relates to a novel two-step warming procedure to warm cryopreserved cells from a cryopreservation temperature. This two-step warming procedure is particularly advantageous for warming cells attached to a fixed substrate.
The two stage method of thawing cells from a cryopreserved state includes first warming the cells from a cryopreservation temperature to a transition temperature of at least xe2x88x9230xc2x0 C. in a first, slow-warming stage by exposing the cells to a first environment having a temperature of less than 30xc2x0 C., and once the cells have obtained the transition temperature, subsequently further warming the cells from the transition temperature by exposing the cells to a second environment having a temperature of at least 32xc2x0 C. in a second, rapid-warming stage. After the cells obtain the transition temperature in the first stage, the cells may be equilibrated at the transition temperature for a period of time prior to conducting the second stage warming. The method is particularly useful in warming cryopreserved cells attached to a substrate, e.g., a fixed substrate.
A heat transfer device in association with the cryopreserved cells may also be used to further assist in the warming procedure, particularly when the cells are attached to a fixed substrate. In this regard, the invention also relates to a warming apparatus for cryopreserved cells, the apparatus including a vessel with which the cryopreserved cells are associated and a heat transfer device in association with the vessel.